Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/70—Carbohydrates; Sugars; Derivatives thereof
  • A61K31/715—Polysaccharides, i.e. having more than five saccharide radicals attached to each other by glycosidic linkages; Derivatives thereof, e.g. ethers, esters
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
  • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof
  • C08B37/006—Heteroglycans, i.e. polysaccharides having more than one sugar residue in the main chain in either alternating or less regular sequence; Gellans; Succinoglycans; Arabinogalactans; Tragacanth or gum tragacanth or traganth from Astragalus; Gum Karaya from Sterculia urens; Gum Ghatti from Anogeissus latifolia; Derivatives thereof
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
  • A61P7/02—Antithrombotic agents; Anticoagulants; Platelet aggregation inhibitors
• • C—CHEMISTRY; METALLURGY
  • C08—ORGANIC MACROMOLECULAR COMPOUNDS; THEIR PREPARATION OR CHEMICAL WORKING-UP; COMPOSITIONS BASED THEREON
  • C08B—POLYSACCHARIDES; DERIVATIVES THEREOF
  • C08B37/00—Preparation of polysaccharides not provided for in groups C08B1/00 - C08B35/00; Derivatives thereof

Definitions

• the present invention relates to novel synthetic polysaccharides having the anticoagulant and antithrombotic pharmacological activities of heparin.
• Heparin belongs to the family of glycosaminoglycans (GAGs), which are Heterogeneous natural sulfated polysaccharides.
• Heparin preparations are mixtures of chains comprising a number monosaccharide units ranging from 10 to 100 and more. To this heterogeneity of size is added a heterogeneity of structure. at the level of the nature of constituent monosaccharides, but also at the level of the substituents which they carry (L. Rodén in: "The Biochemistry of Glycoproteins and Glycosaminoglycans", Ed. By Lennarz W.J., Plenum Press, New York and London, 267-371, 1980).
• Each family of natural GAGs generally has a range of activities pharmacological. All are brought together in the preparations that can be obtained at from natural products. So, for example, heparins and heparan sulfate have antithrombotic activity which is linked to the simultaneous action on several coagulation factors.
• Heparin catalyzes, in particular via antithrombin III (AT III), the inhibition of two enzymes which intervene in the blood coagulation cascade, namely, factor Xa and factor IIa (or thrombin).
• AT III antithrombin III
• Preparations of low molecular weight heparins (LMWH) contain chains formed from 4 to 30 monosaccharides and have the property of acting more selectively on factor Xa than on thrombin.
• Certain synthetic oligosaccharides in particular those described in EP 84999, have the property of selectively inhibiting, via antithrombin III, factor Xa without any activity on thrombin.
• DLA antithrombin binding domain
• DLT less well defined binding domain
• the synthetic oligosaccharides corresponding to the DLA domain of heparin are known and manifest antithrombotic activity in venous thrombosis. These compounds are described in EP 529715, EP 621282 and in the Canadian patent 2040905.
• new pofysaccharide derivatives can be synthesized relatively simply and are biologically active. They are in particular anticoagulants and antithrombotic.
• these polysaccharides due to the synthesis of these polysaccharides, it is possible to selectively modify their structure, and in particular to eliminate unwanted sulphate substituents involved in the interaction with certain proteins.
• polysaccharides can be obtained which are powerful antithrombotic and anticoagulant agents and which, moreover, can escape in vivo from the action of proteins such as platelet factor 4 (FP4), which neutralize the effect of the heparin especially on thrombin.
• FP4 platelet factor 4
• sulfated and alkylated polysaccharides can be potent antithrombotics and anticoagulants depending on disposition alkyl groups and sulphate groups carried by the carbohydrate backbone.
• the present invention relates to a new synthetic polysaccharide, comprising a binding domain to antithrombin III consisting of a chain of five monosaccharides carrying a total of two carboxylic acid functions and at least four sulphate groups, this domain being linked directly at its non-reducing end by a thrombin-binding domain comprising a chain of 10 to 25 monosaccharide units chosen from hexoses, pentoses or deoxy sugars in which all the hydroxyl groups are independently etherified by a group ( C 1 -C 6 ) alkyl or estadosftés in sulphate form, as well as its salts, in particular those pharmaceutically acceptable.
• a binding domain to antithrombin III consisting of a chain of five monosaccharides carrying a total of two carboxylic acid functions and at least four sulphate groups, this domain being linked directly at its non-reducing end by a thrombin-binding domain comprising a chain of 10 to 25 monosaccharide units
• the invention relates to a polysaccharide as defined above, characterized in that all of its hydroxyl groups are methylated or esterified under sulfate form and its salts, especially those which are pharmaceutically acceptable.
• a wavy line denotes a bond located either below or above the plane of the cycle pyranose.
• the monosaccharides contained in Po can be the same or different from each other of the others, the interglycosidic bonds can be of the ⁇ or ⁇ type.
• EP 649854 as well as in the literature. They are obtained from synthons also described in the literature according to C. Van Boeckel, M. Petitou, Angew. Chem. Int. Ed. Engl., 1993, 32, 1671-1690.
• these pentasaccharides are prepared using new building blocks which constitute a further aspect of the invention.
• the present invention relates to intermediaries new useful for the preparation of compounds (I).
• the polysaccharide part Po can consist of 10 to 25 units alkylated and di- or trisulfated monosaccharides.
• the polysaccharide part Po can consist of 10 to 25 units alkylated and mono- or disulphated monosaccharides.
• the polysaccharide part Po can consist of 10 to 25 units uncharged and / or partially charged alkylated monosaccharides and / or fully charged.
• Charged or uncharged units may be dispersed throughout the chain or they can on the contrary be grouped into saccharide domains loaded or not loaded.
• the bonds can be 1,2; 1.3; 1.4; 1.5; 1.6; and of type ⁇ or ⁇ .
• L-iduronic acid can be of conformation 1 C 4 2 S 0 or 4 C 1 .
• Advantageous compounds are the salts whose anion corresponds to formula (1.1): in which t represents 5, 6 or 7, and the cation is a pharmaceutically acceptable monovalent cation, as well as the corresponding acids.
• the salts whose anion corresponds to formula (I.2) are also advantageous: in which t represents 5, 6 or 7 and the cation is a monovalent pharmaceutically acceptable cation, as well as the corresponding acids.
• the salts whose anion has the formula (1.3) are particularly advantageous: wherein m represents 1,2 or 3 and t represents 2, 3, 4 or 5, and the cation is a pharmaceutically acceptable monovalent cation, as well as the corresponding acids.
• the preferred salts of the invention are those whose cation is chosen from the cations of alkali metals and more preferably still those whose cation is Na + or K + .
• the present invention also relates to a process for the preparation of compounds of formula (I) characterized in that: in a first step, a completely protected precursor of the desired polysaccharide (I), containing a protected precursor of the Pe domain (this domain being shown in FIGURE 1) extended at its non-reducing end by a precursor protected from the polysaccharide sulfated Po is synthesized then, in a second step, the charged groups negatively are introduced and / or unmasked.
• the synthesis of these precursors of Pe is carried out as indicated previously in from synthons described in the literature or forming part of the present invention.
• the synthesis of the precursor polysaccharide part of Po is carried out according to reactions well known to those skilled in the art, using the methods of synthesis oligosaccharides (G.J. Boons, Tetrahedron, 1996, 52, 1095-1121) or a oligosaccharide when a glycosidic linker donor oligosaccharide is coupled with a glycosidic binding acceptor oligosaccharide to lead to another oligosaccharide whose size is equal to the sum of the sizes of the two species reactive.
• FIGURE 2 A preferred method for the preparation of Po precursors according to the present invention is shown in FIGURE 2 below.
• FIGURE 2 Synthesis of the protected precursor of DLT
• temporary By temporary is meant a substituent kept for a limited number of stages, by semi-permanent a substituent kept for a greater number of stages and by permanent, a substituent kept until the end of the synthesis; the permanent substituents are eliminated in the last step.
• Some groups perms can be part of the final molecule.
• (a) represents a binding donor monosaccharide glycoside in which Z is a temporary protective group of a function hydroxyl, and Y is an anomeric carbon activator, Tn identical or different, are temporary, semi-permanent or permanent substitutes for all other hydroxyl functions.
• Compound (b) which has an unsubstituted hydroxyl group represents a glycosidic acceptor monosaccharide in which identical or different Tn are temporary semi-permanent or permanent substituents of the hydroxyl groups.
• T 1 is a temporary semi-permanent or permanent protective grouping of the anomeric position. It is eliminated when one wants to activate the anomeric carbon.
• the glycosidic binding donor (a) and the glycosidic binding acceptor (b) react together to give the disaccharide (c).
• the disaccharide (c) obtained above is specifically converted into a glycosidic donor donating disaccharide (d) by elimination of T 1 and introduction of and / or into a glycosidic binding acceptor (e) by elimination of Z.
• glycosidic binding donor (d) and the glycosidic binding acceptor (e) react together to give the tetrasaccharide (f) in which t represents 1,
• the compounds like (f) and (g) are converted into glycosidic binding donors, and coupled to the non-reducing terminal unit of completely protected precursors of Pe.
• the terminal unit oligosaccharide is not reducing a glycosidic donor polysaccharide (g) can constitute a part of Pe, in the case where (g) is coupled to the non-reducing terminal unit of a fully protected oligosaccharide which is the precursor of the rest of the structure of Pe.
• the compounds of the invention can naturally be prepared using different strategies known to those skilled in the art of synthesizing oligosaccharides.
• the precursor of the part of the pentasaccharide Pe when W represents an oxygen atom and R 1 a is R 1 is prepared according to the methods of the synthesis of oligosaccharides and particularly according to the methods described in patents EP 84999, EP 301618, EP 454220 and EP 529715 and in patent applications EP 9304769 and EP 94202470.
• complete protection is achieved, it is possible, using the appropriate protective groups, to obtain a free hydroxyl group at position 4 of the non-reducing terminal unit (D).
• the fully protected precursor of Pe is then coupled to this position using known methods of the synthesis of oligosaccharides.
• the pentasaccharide Pe in which W represents a carbon atom and R 1 a is R 1 of formula: in which R and R 1 are as defined for (I) is obtained from the synthon of formula: in which T 1 , T n, which are identical or different, represent a temporary, semi-permanent or permanent substituent, Z is a protecting group for a hydroxyl function itself obtained by a synthesis carried out by means of a radical reaction between a generating monosaccharide of free radicals and a monosaccharide comprising a double bond, the C-disaccharide thus obtained being then converted into synthon (II.1) according to the conventional methods described above according to C. Van Boeckel, M. Petitou.
• the pentasaccharide Pe in which there is a substituent R 1 a which constitutes a unit of L-iduronic acid of locked configuration of formula: in which R and R 1 are as defined for (I) and W represents an oxygen atom are obtained from the synthon of formula: in which T 1 , T n, which are identical or different, represent a temporary, semi-permanent or permanent substituent, Z is a protective group for a hydroxyl function itself obtained by a synthesis carried out according to the methods described in the literature MK Gurjar et al ., Tetrahedron letters, 1995, 36, 11, 1937-1940, 1933-1936 and 1994, 35, 14, 2241-2244.
• Intermediaries (II.1) and (III.1) are particularly new intermediaries useful for the preparation of the compounds (I) according to the invention.
• the pentasaccharides Pe can therefore be obtained from these synthons disaccharides (II.1) or (III.1), as described in the publication of C.A.A Van Boeckel and M. Petitou, Angew. Chem. Int. Ed. Engl. cited above.
• semi-permanent groups used above is meant groups eliminable first after glycosylation reactions when the skeleton carbohydrate contains the number of desired motifs, without removing or altering the other groups present, allowing the introduction of functional groups desired positions they occupy.
• Permanent groups are groups capable of maintaining the protection of OH functions during the introduction of functional groups to the places semi-permanent groups.
• These groups are chosen from those compatible with functional groups introduced after elimination of the semi-permanent groups. It is also about groups inert with respect to the reactions carried out for the establishment of these functional groups and which can be eliminated without these functional groups are altered.
• the permanent groups are preferably the C 1 -C 6 alkyl groups.
• benzyl and acetyl groups As an example of a semi-permanent and / or temporary group, mention may be made of benzyl and acetyl groups, levulinyl, p-methoxybenzyl, etc.
• the substituents in position 3 of the uronic units of the target compound may already be present in the starting building blocks, as well as the substituent R 1 .
• Protecting groups used in the process for the preparation of compounds (I) are those commonly used in sugar chemistry for example in Protective Groups in Organic Synthesis, TW Greene, John Wiley & sons, New-York, 1981.
• the protecting groups are advantageously chosen, for example, from acetyl, halogenomethyl, benzoyl, levulinyl, benzyles, benzyles groups substituted, optionally substituted trityles, tetrahydropyranyl, allyles, pentenyles, tert-butyldimethylsilyles (tBDMS) or trimethylsilylethyls
• acetyl, halogenomethyl benzoyl, levulinyl, benzyles, benzyles groups substituted, optionally substituted trityles, tetrahydropyranyl, allyles, pentenyles, tert-butyldimethylsilyles (tBDMS) or trimethylsilylethyls
• tBDMS tert-butyldimethylsilyles
• the activating groups are those conventionally used in sugar chemistry according to for example G.J. Boons, Tetrahedron, 1996, 52, 1095-1121. These activating groups are chosen for example from imidates, thioglycosides, pentenylglycosides, xanthates, phosphites or halides.
• the compounds of the invention in the form of acids can then be neutralized by a base to obtain a desired salt.
• any base can be used mineral or organic, with the compounds of formula (I), salts pharmaceutically acceptable.
• sodium hydroxide, potassium hydroxide are used as the base.
• calcium or magnesium are used as the base.
• the sodium and calcium salts of the compounds formula (I), are the preferred salts.
• the protective groups used are those usually used by a person skilled in the art in sugar chemistry, for example according to EP 84999 or again according to Protective Groups in Organic Synthesis, TW Greene, J. Wiley & sons, 1995.
• the compounds (I) thus obtained can be optionally salified.
• the compounds of formula (I) above also include those in which one or more hydrogen or carbon atoms have been replaced by their isotope radioactive, for example tritium or carbon-14.
• isotope radioactive for example tritium or carbon-14.
• Such labeled compounds are useful in research, metabolism or pharmacokinetics, in biochemical tests as ligands.
• the compounds according to the invention have been the subject of biochemical studies and pharmacological studies which have shown that they have very interesting properties.
• the compounds of the present invention which selectively bind AT III with a affinity equal to or greater than that of heparin, have the properties anticoagulants and antithrombotics of heparin.
• the overall antithrombotic activity of the products of formula (I) was evaluated intravenously or subcutaneously in the rat, in a model of venous stasis and induction by thromboplastin, according to the method described by J. Reyers et al . in Thrombosis Research, 1980, 18 , 669-674 as well as in an arterial thrombosis model consisting of a shunt implanted between the carotid artery and the jugular vein of rats as described by Umetsu et al. Thromb. Haemost., 1978, 39, 74-83.
• the ED 50 of the compounds of the invention is at least of the same order or lower than that of the other synthetic heparinoids already known (ED 50 between 5 and 500 ⁇ g / kg).
• the compounds of the invention therefore exhibit a specificity of action and a particularly advantageous anticoagulant and antithrombotic activity.
• the compounds of the present invention are very interesting drugs. Their toxicity is perfectly compatible with this use. They are also very stable and are therefore particularly suitable for constituting the active ingredient in specialties pharmaceuticals.
• the compounds of the invention are not neutralized by high doses of platelet cationic proteins such as platelet factor 4 (FP4) released during activation of these during the thrombosis process.
• FP4 platelet factor 4
• thromboembolic disorders associated with atherosclerosis and diabetes such as unstable angina, stroke cerebral, restenosis after angioplasty, endarterectomy, fitting of prostheses endovascular; or thromboembolic disorders associated with rethrombosis after thrombolysis, infarction, ischemic dementia, arterial disease peripheral, hemodialysis, atrial fibrillations or during the use of aorto-coronary bypass vascular prostheses.
• thromboembolic disorders associated with thrombosis after thrombolysis, infarction, ischemic dementia, arterial disease peripheral, hemodialysis, atrial fibrillations or during the use of aorto-coronary bypass vascular prostheses can also be used for the treatment or prevention of pathologies venous thromboembolism such as pulmonary embolism.
• the compounds of the present invention can cover prostheses and thus make them hemocompatible.
• they can be attached to intravascular prostheses (stents).
• they can possibly be chemically modified by introduction to the non-reducing or reducing end of a suitable arm, as described according to EP 649 854.
• the compounds of the present invention can also be used as adjuvant during endarterectomy performed with porous balloons.
• the compounds of the invention are very stable and are therefore particularly suitable for constituting the active principle of medicinal products.
• the subject of the present invention is therefore a pharmaceutical composition containing, as active ingredient, a polysaccharide synthesis as defined above.
• the invention preferably relates to pharmaceutical compositions containing as active principle, a compound of formula (I), (I.1), (I.2), (I.3) or one of its salts pharmaceutically acceptable, optionally in combination with one or more inert and suitable excipients.
• each dosage unit the active ingredient is present in the appropriate quantities at the daily doses envisaged.
• each dosing unit is suitably adjusted according to the dosage and the type of administration planned, for example example tablets, capsules and the like, sachets, ampoules, syrups and the like, drops, transdermal or transmucosal patch so that such a unit of dosage contains 0.1 to 100 mg of active ingredient, preferably 0.5 to 50 mg.
• the compounds according to the invention can also be used in combination with a other active ingredient useful for the desired therapy such as for example antithrombotics, anticoagulants, antiplatelet agents such as by example dipyridamole, aspirin, ticlopidine, clopidogrel or antagonists of glycoprotein IIb / IIIa complex.
• a other active ingredient useful for the desired therapy such as for example antithrombotics, anticoagulants, antiplatelet agents such as by example dipyridamole, aspirin, ticlopidine, clopidogrel or antagonists of glycoprotein IIb / IIIa complex.
• compositions are formulated for administration to mammals, including humans, for the treatment of the aforesaid diseases.
• the pharmaceutical compositions thus obtained are advantageously presented in various forms, such as for example, injectable solutions or drinkable, dragees, tablets or capsules.
• injectable solutions are the forms preferred pharmaceuticals.
• the pharmaceutical compositions of this invention are particularly useful for the treatment as a preventive or curative, vascular wall disorders, such as atherosclerosis, hypercoagulability states observed for example following surgical operations, development tumor or coagulation disturbances, induced by bacterial activators, viral, or enzymatic.
• the dosage can vary widely depending on age, weight and state of health of the patient, nature and severity of the condition, as well that of the route of administration. This dosage includes the administration of one or several doses of approximately 0.1 mg to 100 mg per day, preferably approximately 0.5 to 50 mg daily, intramuscularly or subcutaneously, in continuous administrations or at regular intervals.
• the present invention therefore also relates to pharmaceutical compositions which contain, as active principle, one of the above compounds, optionally in association with another active ingredient. These compositions are produced so as to be able to be administered by the digestive or parenteral route.
• the active ingredient can be administered in the form administration units, mixed with conventional pharmaceutical carriers, animals and humans.
• Appropriate unit forms of administration include oral forms such as tablets, capsules, powders, granules and oral solutions or suspensions, forms sublingual and oral administration, subcutaneous administration forms, intramuscular, intravenous, intranasal or intraocular and the forms rectal administration.
• a solid composition When a solid composition is prepared in the form of tablets, it is mixed the main active ingredient with a pharmaceutical vehicle such as gelatin, starch, lactose, magnesium stearate, talc, gum arabic or like.
• a pharmaceutical vehicle such as gelatin, starch, lactose, magnesium stearate, talc, gum arabic or like.
• a preparation in capsules is obtained by mixing the active ingredient with a diluent and by pouring the mixture obtained into soft or hard capsules.
• Water dispersible powders or granules may contain the active ingredient in admixture with dispersing agents or wetting agents, or agents suspension, such as polyvinylpyrrolidone, as well as with sweeteners or flavor correctors.
• suppositories are used which are prepared with binders that melt at the rectal temperature, for example cocoa butter or polyethylene glycols.
• aqueous suspensions for parenteral, intranasal or intraocular administration, aqueous suspensions, isotonic saline solutions or sterile solutions and injectables which contain dispersing agents and / or wetting agents pharmacologically compatible, for example propylene glycol or butylene glycol.
• the active principle can be formulated in the presence a promoter such as a bile salt, a hydrophilic polymer such as for example hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxyethylcellulose, ethylcellulose, carboxymethylcellulose, dextran, polyvinylpyrrolidone, pectins, starches, gelatin, casein, acrylic acids, esters acrylics and their copolymers, vinyl polymers or copolymers, alcohols vinyl, alkoxypolymers, polyethylene oxide polymers, polyethers or their mixture.
• a promoter such as a bile salt
• a hydrophilic polymer such as for example hydroxypropylcellulose, hydroxypropylmethylcellulose, hydroxyethylcellulose, ethylcellulose, carboxymethylcellulose, dextran, polyvinylpyrrolidone, pectins, starches, gelatin, casein, acrylic acids, esters acrylics and their copolymers, vinyl polymers or
• the active principle can also be formulated in the form of microcapsules, possibly with one or more carriers or additives.
• the active ingredient can also be presented in the form of a complex with a cyclodextrin, for example ⁇ , ⁇ or ⁇ cyclodextrin, 2-hydroxypropyl- ⁇ -cyclodextrin or methyl- ⁇ -cyclodextrin.
• a cyclodextrin for example ⁇ , ⁇ or ⁇ cyclodextrin, 2-hydroxypropyl- ⁇ -cyclodextrin or methyl- ⁇ -cyclodextrin.
• the active ingredient can also be released by a balloon containing it or by a endovascular expander introduced into the blood vessels. The effectiveness pharmacological of the active ingredient is thus not affected.
• Subcutaneous administration is the preferred route.
• a solution of tert- butyldimethylsilyl triflate in dichloromethane (1M, 0.2 mol / mol of imidate) is added, under argon at ⁇ 20 ° C., to a solution of the imidate and of the glycosyl acceptor in dichioromethane (17.5 mL / mmol) in the presence of 4 ⁇ molecular sieve. After 10-20 minutes (TLC), solid sodium hydrogen carbonate is added. The solution is filtered, washed with water, dried and evaporated to dryness.
• the compound to be deprotected is dissolved in a 2: 1 ethanol / toluene mixture. (42 mL / mmol) and hydrazine acetate (5 mol / mol) is added. Let stir for 15-30 minutes (TLC) and concentrate.
• the thioglycoside and the glycosyl acceptor are dissolved in an inactinic glass flask in anhydrous toluene (18 ml / mmol of thioglycoside) in the presence of 4 ⁇ molecular sieve.
• the mixture is stirred for 1 hour at room temperature. Cool to 0 ° C. and add N- iodosuccinimide (3 mol / mol of thioglycoside) then silver triflate (0.28 mol / mol of thioglycoside). After 10-15 minutes (TLC), solid sodium hydrogen carbonate is added. After filtration, the solution is washed with a 1M aqueous sodium thiosulfate solution, water, dried and evaporated.
• esters A 5M aqueous sodium hydroxide solution is added (in an amount such that the concentration of sodium hydroxide is 0.5 M at the end of the addition) to a solution of an ester in methanol (150 ml / mmol ). After 2-5 hours, water is introduced and it is passed through a column of Sephadex® G-25 gel (1.6 x 115 cm) eluted with water. Concentrate, pass through a Dowex® 50 H + column (2 mL) and lyophilize. At this stage, it is verified by 1 H NMR that all the protective groups have been removed. If necessary, the product is again subjected to hydrogenation and / or saponification.
• Triethylamine / sulfur trioxide complex (5 mol / mol hydroxyl function) is added to a solution in dimethylformamide (5 mg / ml) of the compound to be sulfated. After one day at 55 ° C., the solution is deposited at the top of a column of Sephadex® G-25 (1.6 x 115 cm) eluted with 0.2 M sodium chloride. The fractions containing the product are concentrated and it is desalinated using the same column eluted by water. The final compound is obtained after lyophilization.
• Trifluoromethanesulfonic acid (1.10 mL, 0.012 mol) is added to a suspension of 3- O- methyl-glucose commercial (135 g, 0.7 mol) in allyl alcohol (1 L). The mixture is heated at 120 ° C for 2 hours. Neutralized by addition of triethylamine (2 mL) and evaporated to dryness. To the above crude compound dissolved in N, N- dimethylformamide (2 L), ⁇ , ⁇ -dimethoxytoluene (136 mL, 0.9 mol) and camphorsulfonic acid (25 g, 0.13 mmol) are added ). The mixture is heated at 80 ° C. for 1 hour under vacuum.
• a solution of trifluoroacetic anhydride is added to a solution of 8 (12.0 g, 33.3 mmol) and triethylsilane (21.3 mL, 133 mmol) in anhydrous dichloromethane (50 mL) at 0 ° C (306 ⁇ L, 2.10 mmol) in trifluoroacetic acid (10 mL).
• the mixture is stirred for 4 hours (TLC), diluted with ethyl acetate and a 1M aqueous solution of sodium hydroxide is added to pH 9. Extraction is carried out with ethyl acetate, washed with water, dried and concentrated.
• 1,5-Cyclooctadiene-bis [methyldiphenyl-phosphine] -iridium hexafluorophosphate is added to a solution of 10 (1.36 g, 1.66 mmol) in tetrahydrofuran without peroxides (4.30 mL).
• the solution is degassed, placed under an argon atmosphere and hydrogen is introduced.
• the mixture is left stirring for 10 minutes (TLC) and evaporated. It is taken up in dichloromethane, washed with a saturated aqueous solution of sodium hydrogencarbonate, with water, dried and concentrated.
• Phenyl 6- O -benzyl-2,3-di- O -methyl-1-thio- ⁇ -D-glucopyranoside (24)
• Methyl iodide (34 mL, 0.536 mol) is added dropwise under argon to a solution of compound 33 (64.1 g) in N , N- dimethylformamide (600 mL). Cool to 0 ° C, and slowly add sodium hydride (13.5 g, 0.536 mol). The suspension is stirred for 2 hours at room temperature, then cooled to 0 ° C., and methanol (35 mL) is added dropwise and after 2 hours of stirring, the mixture is diluted in acetate. ethyl (500 mL) and water (600 mL). The aqueous phase is extracted with ethyl acetate, the organic phases are washed with water, dried, and concentrated.
• Triethylamine (65 mL, 0.47 mol) and acetic anhydride (89 mL, 0.94 mol) are added dropwise and at 0 ° C. to a suspension of 53 (47.22 g, 93, 6 mmol) in dichloromethane (450 mL).
• 4-dimethylaminopyridine (5.71 g, 46.8 mmol) is then added and the mixture is left to stir for 1.5 hours at room temperature.
• the reaction is stopped by adding methanol (45 mL, 1.12 mol) and then washed successively with a cold aqueous 10% solution of potassium hydrogen sulphate, water, a saturated solution of sodium hydrogen carbonate, and some water.
• a suspension of 54 (25.0 g, 35.0 mmol), borane-trimethylamine complex (20.4 g, 0.28 mol) and molecular sieve (33 g, 4 ⁇ ) are stirred for 1 hour under argon in toluene (810 mL). Cool to 0 ° C and slowly add aluminum chloride (14.0 g, 0.11 mol). The mixture is stirred for 25 minutes (TLC), the reaction medium is poured into a cold aqueous solution of 20% potassium hydrogen sulfate and stirred for 1 hour at 0 ° C., then filtered (Celite). The organic phase is washed with water, with a 2% aqueous solution of sodium hydrogencarbonate, with water, dried and concentrated.
• Acetic anhydride (1.47 mL, 15.5 mmol) is added at 0 ° C to a mixture of 55 (5.6 g, 7.77 mmol), triethylamine (1.19 mL, 8 , 54 mmol), and 4-dimethylaminopyridine (190 mg, 1.55 mmol) in dichloromethane (40 mL). After 40 minutes of stirring (TLC) at room temperature, diluted with dichloromethane (50 mL) and washed with a cold aqueous solution of 10% potassium hydrogen sulfate, water, saturated hydrogen carbonate solution sodium, and water.
• the glycosyl acceptor 26 (220 mg, 0.16 mmol) and imidate 72 (344 mg, 0.16 mmol) are dissolved in a dichloromethane / ethyl ether mixture 1: 2 (5 mL). 4 ⁇ molecular sieve (750 mg / mmol) is added and the mixture is left stirring at 25 ° C. for 1 hour. The mixture is cooled to -25 ° C. and a 1M solution of tert- butyldimethylsilyl triflate in dichloromethane (0.20 mol / mol of imidate) is added. The mixture is left stirring for 15 minutes and then solid sodium hydrogencarbonate is added, filtered and concentrated.
• glycosyl acceptor 74 (112 mg, 33.9 ⁇ mol) and the imidate 17 (59.1 mg, 37.2 ⁇ mol) (see PREPARATION 16) are dissolved in toluene (2 mL). 4 ⁇ molecular sieve (42 mg) is added and the mixture is left stirring at 25 ° C. for 1 hour. The mixture is cooled to -20 ° C. and a 1M solution of tert- butyldimethylsilyl triflate in toluene (0.20 mol / mol of imidate) is added. The mixture is left stirring for 15 minutes and then solid sodium hydrogencarbonate is added, filtered and concentrated.
• reaction mixture is diluted with dichloromethane (100 mL), washed with a saturated aqueous solution of sodium hydrogencarbonate, dried (magnesium sulfate), filtered, and concentrated.
• the crude compound 83 (8.17 g, 68% from 81 ) is directly involved in the deacetylation reaction.
• Compound 100 is transformed into compound 101 according to the same procedure as that described for the preparation of compound 96 .
• TLC: Rf 0.50, silica gel, dichloromethane / methanol / 9/1 v / v
• Compound 110 is transformed into compound 111 according to the same procedure as that described for the preparation of compound 96.
• TLC: Rf 0.50, silica gel, toluene / ethyl acetate / ethanol 6/2/2 v / v / v
• the product is first purified by chromatography on Sephadex LH 20 (dichloromethane / methanol 1/1 v / v) followed by chromatography on a column of silica gel to give 124 mg of compound 141 in ⁇ / ⁇ ratio of 8 / 2.
• TLC: Rf 0.60, silica gel, toluene / acetone 1/1 v / v
• Compound 141 is transformed into compound 142 according to the procedure described for the preparation of compound 140.
• Thioglycoside 147 (105 mg, 0.066 mmol) and acceptor 142 (55 mg, 0.017 mmol), ( ⁇ / ⁇ of 8/2) are coupled according to the procedure described for compound 109.
• the product is first purified by chromatography on Sephadex LH 20 (dichloromethane / methanol 1/1) followed by chromatography on a column of silica gel (diethyl ether / ethyl acetate / ethanol 9 / 0.5 / 0.5 v / v / v ) to give 49 mg of compound 148.
• TLC: Rf 0.30, silica gel, diethyl ether / ethyl acetate / ethanol 85 / 7.5 / 7.5 / v / v / v v v v v v v )
• Diol 151 is taken up (10 g, 42.7 mmol) in anhydrous dichloromethane (100 ml) and tert-butyldimethylsilyl chloride (7.1 g 47.3 mmol) and imidazole (5.8) are added. g, 85.3 mmol).
• the reaction mixture is stirred at room temperature. After 2 hours the mixture is diluted in dichloromethane and washed with water. The organic phase is dried over magnesium sulphate, concentrated and the residue is purified by chromatography on a column of silica gel (ethyl acetate / cyclohexane 1/9 vlv) in order to obtain the desired product 152 (11.9 g 80%) in the form of a syrup. [ ⁇ ] D - 34 ° (c 1.9, CHCl 3 ).
• Oxalyl chloride (3.2 ml, 36.8 mmol) and dimethyl sulfoxide (5.2 ml, 73.4 mmol) are added at -78 ° C. in anhydrous dichloromethane (40 ml) and the mixture is stirred for 30 minutes. Then, compound 152 (6.4 g, 18.4 mmol) is added and the mixture is stirred for a further 1 hour. Then, triethylamine (15.3 ml, 110.0 mmol) is added and after 30 minutes, the reaction mixture is diluted in dichloromethane.
• a conventional treatment makes it possible to obtain the compound 5-ulose ( 153 ) which is directly used for the following reaction.
• the crude ketone 153 is taken up in anhydrous tetrahydrofuran (100 ml) and a 1M solution of vinyl magnesium bromide in tetrahydrofuran (28 ml, 27.6 mmol) is added at O ° C. After 1 hour, the reaction mixture is diluted not with ammonium chloride and washed with water. The organic phase is dried over magnesium sulfate, concentrated and the residue is purified by chromatography on a column of silica gel (ethyl acetate / cyclohexane 1/9 v / v) to obtain the desired compound 154 (70% , 4.8 g) in the form of a syrup. [ ⁇ ] D - 40 ° (c 1.3, CHCl 3 ). Anal. Calculated: C, 57.72, H, 9.15. Found: C, 57.77, H, 9.23.
• Ketone 161 is taken up in anhydrous tetrahydrofuran (15 ml) and a 1N solution of super hydride in tetrahydrofuran (4 ml, 4.0 mmol) is added at -78 ° C. The reaction mixture is stirred at room temperature for 1 hour and then added 5% sodium hydroxide (2 ml) and hydrogen peroxide (1 ml). The solvent is evaporated off and the residue is taken up in ethyl acetate and washed with water. The organic phase is dried over magnesium sulphate, concentrated and the residue is purified by chromatography (ethyl acetate / cyclohexane 2/1 v / v) to obtain compound 162 (1.0 g, 70%). [ ⁇ ] D - 11 ° (c 0.5, CHCl 3 ). CI-MS: 724 (M + 18), 707 (M + 1).
• the suspension is vigorously stirred at room temperature overnight, diluted with water and extracted with ethyl acetate.
• the organic phase is dried over magnesium sulphate, concentrated and then we go directly to the next reaction.
• the crude acid is taken up in dimethylformamide (25 ml) and tetrabutylammonium iodide (0.7 g, 2.0 mmol), potassium bicarbonate (0.25 g, 2.5 mmol) are added and benzyl bromide (0.250 ml, 2.1 mmol).
• the reaction mixture is stirred at room temperature for 5 hours.
• the reaction mixture is extracted with water and ether.
• the completely deprotected hexadecasaccharide 150 (26 mg, 0.0084 mmol) is dissolved in dimethylformamide (0.87 ml). Under a nitrogen atmosphere, a triethylamine sulfide trioxide complex (125 mg, 0.67 mmol, 80 eq) is added and the mixture is stirred for 16 hours at 50 ° C. The mixture is cooled to 0 ° C and add aqueous sodium hydrogencarbonate (227 mg, 2.6 mmol). The mixture is concentrated to a small volume and applied to a column of Sepahdex G-25, eluted with water / acetonitrile 9/1 vlv.

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